Journal: Cell Communication and Signaling : CCS

Article Title: ATXN3 promotes prostate cancer progression by stabilizing YAP

doi: 10.1186/s12964-023-01073-9

Figure Lengend Snippet: ATXN3 depletion decreases Hippo signaling activity in prostate cancer cells. A The siRNAs specific to each deubiquitinating enzyme were transfected into LnCap cells. After 48 h, cells were lysed and the YAP protein level was analyzed by Western blot. B , C ATXN3 depletion decreased YAP protein level. D , E ATXN3 depletion decreased YAP target genes using two different siRNA oligos. F ATXN3 depletion decreased TOP-luciferase activity. LnCap and C4-2B cells were transfected with SiATXN3 or SiControl together with YAP/TEAD-luciferase reporter plasmid. Luciferase activity was measured 48 h after transfection. G ATXN3 and YAP was upregulated in PC samples. H ATXN3 correlated YAP in human PC samples. Results shown are representative of 3 independent experiments. Data are represented as mean ± SD of biological triplicates.* P value < 0.05; ** P value < 0.01; *** P value < 0.001 by unpaired, 2-tailed Student’s t tests.

Article Snippet: The human prostate cancer cell lines LnCap, C4-2B and human embryonic kidney HEK293T cells purchased from the Procell Life Science&Technology Co,, Ltd (China).

Techniques: Activity Assay, Transfection, Western Blot, Luciferase, Plasmid Preparation

Journal: Cell Communication and Signaling : CCS

Article Title: ATXN3 promotes prostate cancer progression by stabilizing YAP

doi: 10.1186/s12964-023-01073-9

Figure Lengend Snippet: ATXN3 associates with YAP. A An immunofluorescence assay demonstrated that ATXN3 and YAP at least partially colocalized in LnCap and C4-2B cells. B Co-IP assay reveals association between endogenous ATXN3 and YAP in LnCap cells. LnCap cells were harvested with RIPA lysis buffer. Co-IP was performed using antibody as indicated. C LnCap cells transfected with Flag-ATXN3 were lysed and the lysates were incubated with GST-YAP or GST protein. The interacted ATXN3 was detected by western blot. D ATXN3 domain structure and deletion mutants used in the study. E The Josephin domain of ATXN3 interacted with YAP. HEK293 cells were transfected with 2 µg Myc-YAP together with Flag-ATXN3 full length or mutants. After 24 h, cells were harvested with NP-40 lysis buffer. Co-IP was performed using Myc antibody. The possible interacted ATXN3 domains were detected by Flag antibody. F . YAP domain structure and deletion mutants used in the study. G The WW domain of YAP interacted with ATXN3. HEK293 cells were transfected with 2 µg Flag-ATXN3 together with Myc-YAP full length or mutants. After 24 h, cells were harvested with NP-40 lysis buffer. Co-IP was performed using Flag antibody. The possible interacted YAP domains were detected by Myc antibody

Article Snippet: The human prostate cancer cell lines LnCap, C4-2B and human embryonic kidney HEK293T cells purchased from the Procell Life Science&Technology Co,, Ltd (China).

Techniques: Immunofluorescence, Co-Immunoprecipitation Assay, Lysis, Transfection, Incubation, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: ATXN3 promotes prostate cancer progression by stabilizing YAP

doi: 10.1186/s12964-023-01073-9

Figure Lengend Snippet: ATXN3 de-polyubiquitylates YAP. A LnCap cells transfected with the indicated siRNA were treated with MG132 for 6 h before collection. YAP was immunoprecipitated with anti-YAP and immunoblotted with anti-HA. B Immunoblotting was used to detect the ubiquitination of YAP in 293 T cells co-transfected with Flag-YAP, HA-Ubiquitin and Myc-ATXN3 (wild type or C14A). C ATXN3 removed the ubiquitin chain of YAP in a dose-dependent manner. D HA-WT, K6, K11, K27, K29, K33, K48 or K63 Ub were co-transfected with Flag-YAP and Myc-ATXN3 into HEK293T cells. After treatment with 10 μM MG132 for 6 h, cell lysates were subjected to ubiquitination assay and the ubiquitination level of YAP detected by HA antibody

Article Snippet: The human prostate cancer cell lines LnCap, C4-2B and human embryonic kidney HEK293T cells purchased from the Procell Life Science&Technology Co,, Ltd (China).

Techniques: Transfection, Immunoprecipitation, Western Blot, Ubiquitin Proteomics

Journal: Cell Communication and Signaling : CCS

Article Title: ATXN3 promotes prostate cancer progression by stabilizing YAP

doi: 10.1186/s12964-023-01073-9

Figure Lengend Snippet: ATXN3 depletion inhibits prostate cancer cell proliferation, invasion stem-like properties. A, B ATXN3 depletion inhibited prostate cancer proliferation. C , D ATXN3 depletion induced G1 cell cycle arrest in prostate cancer cells. E ATXN3 depletion decreased clone formation capability of prostate cancer cells. F , G Representative images of EdU assay of prostate cancer cells. H Tranwell invasion assay of prostate cancer cells. I ATXN3 depletion decreased sphere formation of HCC cells. J ATXN3 depletion inhibits the tumor growth in vivo. C4-2B cells were stably transfected with lentivirus carrying a scrambled shRNA or ATXN3 shRNA. 1 × 10 6 LnCap cells were injected to the right dorsal flank of each mouse. Tumor sizes were measured every 5 days until the end of the experiment. K Representative images of immunohistochemical staining for Ki67, ATXN3 and YAP. (L). ATXN3 depletion suppressed the lung metastasis of ATC in mice. 0.5 × 10 6 C4-2B cells were intravenously injected into each mouse through the tail vein (n = 6). The lungs were harvested 4 weeks after injection. Results shown are representative of 3 independent experiments. Data are represented as mean ± SD of biological triplicates.* P value < 0.05; ** P value < 0.01; *** P value < 0.001

Article Snippet: The human prostate cancer cell lines LnCap, C4-2B and human embryonic kidney HEK293T cells purchased from the Procell Life Science&Technology Co,, Ltd (China).

Techniques: EdU Assay, Invasion Assay, In Vivo, Stable Transfection, Transfection, shRNA, Injection, Immunohistochemical staining, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: ATXN3 promotes prostate cancer progression by stabilizing YAP

doi: 10.1186/s12964-023-01073-9

Figure Lengend Snippet: Increased YAP expression reverses the effect induced by ATXN3 depletion. A Cell proliferation assay of LnCap. B Clone formation assay of LnCap. C Representative images of EdU assay of LnCap. D Transwell invasion assay of LnCap. E Sphere formation assay of LnCap. F Overexpression of YAP in ATXN3-knockdown cells partly recovered tumor growth and lung metastasis in vivo. 1 × 10 6 C4-2B cells transfected with indicated plasmids were injected to the right dorsal flank of each mouse (n = 6). Tumor sizes were measured every 5 days until the end of the experiment. G For lung metastasis analysis, 0.5 × 10 6 C4-2B cells were intravenously injected into each mouse through the tail vein (n = 6). The lungs were harvested 4 weeks after injection. Results shown are representative of 3 independent experiments. Data are represented as mean ± SD of biological triplicates.* P value < 0.05; ** P value < 0.01; *** P value < 0.001

Article Snippet: The human prostate cancer cell lines LnCap, C4-2B and human embryonic kidney HEK293T cells purchased from the Procell Life Science&Technology Co,, Ltd (China).

Techniques: Expressing, Proliferation Assay, Tube Formation Assay, EdU Assay, Transwell Invasion Assay, Over Expression, Knockdown, In Vivo, Transfection, Injection